The mechanism of action of nucleoside analogs was investigated in human carcinoma cell line HT-29 and human promyelocytic leukemia cell line HL-60. Cyclopentenyl adenosine (neplanocin A) was an effective inhibitor of RNA and DNA methylation in HL-60 cells via its metabolism to an S-adenosylmethionine (AdoMet)-like metabolite. However, neplanocin proved to be a poor inducer of differentiation in HL-60 cells as assessed by nitroblue tetrazolium reduction and cellular myc RNA expression. The 3-deaza analog of neplanocin, c3Nep, was as effective as an inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase as neplanocin but possessed 1/10 the cytotoxicity, did not effectively inhibit RNA methylation and was not metabolized to an AdoMet-like metabolite. c3Nep was was ineffective as a differentiating agent in HL-60 cells. The cyclopentenyl cytidine analog (cCyd) possessed a potent inhibitory effect on CTP synthesis and subsequently, DNA synthesis with moderate cytotoxicity against HT-29 and HL-60 cells. However, this agent proved to be a very effective differentiating agent for HL-60 cells, probably as a result of its S phase specific effects at noncytotoxic concentrations. In other studies, the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), was found to produce complete remissions in hairy cell leukemia in phase II clinical trials. Experimental studies in mouse lymphocytes indicated that the antileukemic effect of dCF may be a result of inhibition of lymphokine synthesis, viz. IL-2 and immune interferon, which are known to be growth factors for hairy cell (B-cell) leukemia cells.